Project Summary/Abstract Reversible chemical modifications on DNA and histones are known to significantly impact mammalian gene expression regulation. Prior to our work, no example of reversible chemical modifications on RNA that could affect gene expression had been shown. We previously discovered the first two RNA demethylases: FTO, a protein associated with human fat mass obesity, and ALKBH5, a protein that affects spermatogenesis in a mouse model. These two proteins belong to the AlkB family iron- and 2-ketoglutarate (2-KG)-dependent dioxygenases and catalyze oxidative demethylation of the most prevalent internal modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). These studies provided the first demonstration of reversible RNA modification, and stimulated the emergence of a new area of biological studies on RNA modifications in gene expression regulation. In the past funding period, we have demonstrated oncogenic roles of both FTO and ALKBH5 in human cancers. We have also discovered the first tRNA demethylase, ALKBH1, which reverses the N1- methyladenosine (m1A) in tRNA to significantly affect translation initiation and elongation. In the current application, we plan to thoroughly characterize the cytoplasmic versus nuclear RNA demethylation by FTO. Our preliminary studies revealed that RNA substrates of FTO can vary dramatically in different cellular compartments. While FTO is generally regarded as a nuclear protein, it can localize to cytoplasm and mediates functionally significant cytoplasmic RNA demethylation. Previous studies have also uncovered intriguing roles of FTO in UV DNA damage response. Its nuclear demethylation activity may alter the chromatin state and affect recruitment of various factors in damage response. We plan to investigate the extent of nuclear versus cytoplasmic demethylation and understand factors that dictate the cytoplasmic location and demethylation of FTO. These new information will be critical in evaluating small molecule inhibitors that target FTO to suppress tumor progression. In addition to mRNA m6A demethylation, we have recently discovered that ALKBH7, a protein significantly affects mammalian energy homeostasis, is a mitochondrial tRNA demethylase that mediates demethylation of mitochondrial tRNA N2,N2-dimethylguanine (m22G) and affects cellular response to oxidizing agents. We will perform detailed biochemical, structural, and functional characterization of this new tRNA demethylation activity, and reveal its potential impacts on mitochondrial activity.